Our goal is to identify molecular mechanisms involved in the regulation of the beta-adrenergic receptor (BAR)-coupled adenylyl cyclase. At least three major mechanisms of receptor regulation have been identified: desensitization, internalization, and down-regulation. Three subtypes, B1AR, B2AR and B3AR, have been cloned, and found to differ in their tissue distribution as well as their ability to be regulated. For the human subtypes, the order is hB2AR >hB1AR >> hB3AR. For example, we found that when stably expressed in baby hamster kidney (BHK) cells, hB2AR undergoes agonist-mediated internalization more rapidly and extensively than hB1AR. Both BAR kinase (BARK) and beta- arrestin (BARR) have been implicated in B2AR internalization. The BARK- phosphorylated B2AR binds BARR which acts as an adapter protein between the receptor and clathrin. We overexpressed either BARK or BARR in the BHK cells. Both increased the rate and extent of B1AR internalization, BARR being more effective than BARK. Their effects on B2AR internalization were more modest. In this regard, cells overexpressing BARR internalized both subtypes at similar rates and extents. To determine whether hB1AR uses the same clathrin-coated endocytosis pathway as hB2AR, we transiently cotransfected BHK cells with one subtype and either: BARR, dominant-negative mutant (DN) of dynamin, or DN-BARR. Whereas BARR increased the internalization of both subtypes, DN-dynamin and DN-BARR blocked internalization. As dynamin is involved in pinching off of clathrin-coated vesicles, we show that hB1AR is internalized via the same pathway as hB2AR. We believe that differences in rate and extent are due to hB1AR being less succeptible to BARK phosphorylation than hB2AR.We previously showed that activation of protein kinase C (PKC) by phorbol esters in rat glioma C6 cells causes a down-regulation of B1AR binding and mRNA by transcriptional repression of the rB1AR gene. C6 cells also express B2AR, and we find that the latter subtype is similarly regulated. Cells exposed to phorbol ester exhibited a time- and dose-dependent down-regulation of B2AR binding that was preceded by a reduction in B2AR mRNA. Both effects did not occur in cells treated with inactive phorbols and were blocked by a specific PKC inhibitor. When we blocked new transcription with actinomycin D, we found that B2AR mRNA turned over at the same rate in control and phorbol ester-treated cells. To do promoter analysis, we made a construct of the 5-flanking region of the rB2AR gene fused to a luciferase reporter gene and transiently transfected C6 cells with it. In preliminary experiemnts, we observed a modest (15%) decrease in reporter activity in phorbol ester-treated cells. Finally, we did western blotting of lysates from control and treated cells using an antipeptide antibody raised against the C-terminal sequence of the rB2AR. The decrease in receptor protein was the same as the loss in receptor binding activity. Thus, activation of PKC leads to a down- regulation of B2AR mRNA followed by a reduction in B2AR. The most likely mechanism is repression of B2AR gene expression by a PKC- activated transcriptional repressor(s). Our results represent a mechanism of cross-talk between cell signaling pathways at the level of receptor gene expression. - beta-adrenergic receptors/adenylyl cyclase/mRNA/ internalization/down-regulation/protein kinase C /gene transcription/beta-arrestin